Roman M. Link
This is a simplified short form of the BOT2 tutorial for the pre-processing and analysis of wood anatomical microtome slides based on GIMP and ImageJ which is meant for in-class use.
A list of useful GIMP shortcuts can be found here: (https://www.gimpusers.com/gimp/hotkeys).
Note that the term CODE in this document is a placeholder that has to be replaced by the unique ID of sample you are working with! In the screenshots in this example, the CODE is CRI_3_010.
All ImageJ results tables can be saved either in ‘Comma Separated Value’ (.csv) or whitespace/tabstop separated format (generated when saving with a .xls extension, but actually just a plain text format). In either case, the output is optimized for US/UK locales, which means that points are used as a decimal separator. In order to process these files on German systems without compatibility issues, it is important to make sure that the system-wide decimal separator is correctly set before starting the analysis.
In German Windows 10, the option to change the decimal separator is well hidden:
Start Menu ➜ Windows-System ➜ Systemsteuerung ➜ Zeit und Region ➜ Region ➜ Formate ➜ Weitere Einstellungen ➜ Dezimaltrennzeichen
To avoid data compatibility problems, make sure the decimal separator is set to “.”. In this case, you will want the grouping symbol (Symbol für Zifferngruppierung) to be a comma instead of the point symbol used in Germany.
If you do not want to change your system settings, you can alternatively export everything in a .csv format and use Excel’s Daten ➜ Text in Spalten menu to manually set field delimitor and decimal separator.
It is generally recommended to create a separate folder for each sample and to keep all files belonging to the sample in this folder. Folders should always be named by sample ID.
CODE.jpgSave the file CODE_GI_cropped_01.jpg (using the “Export as” option or via Ctrl + Shift + E).
Close tab with original image (do not save changes!)
[optionally] adjust brightness and contrast using color curves (German: Farben ➜ Kurven, English: Colors ➜ Curves)
CODE_GI_cropped_02.jpg.CODE.jpg) with ImageJ (either by the File dialog or by dragging and dropping onto the ImageJ window).
CODE_GI_cropped_02.jpg with ImageJ (drag & drop onto ImageJ bar) - if the scale is correctly set, the dimensions of the picture in µm should be visible in the upper left corner (if the values is in pixels).CODE_GI_cropped_02_TH_01.jpg
.tiff formatCODE_GI_cropped_02_TH_01_Area.xls. The area of the analyzed wood portion can then be calculated by substracting the black area from the total area of the image.Save the image without the black area as CODE_GI_cropped_02_TH_02.jpg
To prepare for automated vessel detection, open the Analyze ➜ Set Measurements dialog and select Area, Shape descriptors, Perimeter, Fit ellipse and Feret’s diameter
.jpg document, specifying the selected Area and Circularity values in the name (e.g. CODE_GI_cropped_02_TH_02_Outlines_300,0.3.jpg) using the File ➜ Save As ➜ Jpeg option in the main menu of ImageJ (not the newly opened Results window!). Make sure the right image window is selected when saving.the simplified workflow in the next couple of screenshots was documented on a computer running Ubuntu Mate and a different version of GIMP, but should work just the same on Windows
CODE_GI_cropped_02_TH_02.jpg) and the outline file(CODE_GI_cropped_02_TH_02_Outlines_300,0.3.jpg) in GIMP (mark both files, right click and select Open with GIMP / Öffnen mit GIMP).CODE_GI_cropped_02_TH_02_edit.jpg using the “Export as” dialog or Strg + Shift + E.Repeat all steps starting from the Analyze Particles section.
Save the new outlines as CODE_GI_cropped_02_TH_02_edit_Outlines_300,0.3.jpg (File ➜ Save As in the main window).
If you are happy with your classification results, save them as CODE_GI_cropped_02_TH_02_edit_Outlines_300,0.3_Results.xls (File ➜ Save as in the Results window).
Use GIMP to copy the new outlines on top of the threshold image and cut out the background (just as in this step).
Save the resulting image (using the “Export as” dialog or _STRG + Shift + E__) using the name CODE_GI_cropped_02_TH_02_edit_Outlines_300,0.3_Analysis.jpg.
Your project folder should now look somewhat like this (note that often, you will have to try more than one setting for circularity and minimum vessels size, and you will have to do more than one edit, which all show up as additional files in the project folder):